Chromogenic plating medium for the rapid presumptive identification of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiesis

ABSTRACT

A plating medium for the presumptive identification of  Bacillus cereus, Bacillus thuringiensis  and/or  Bacillus anthrasis  that includes a nutrient base to facilitate growth of  Bacillus cereus, Bacillus thuringiensis  and  Bacillus anthrasis,  a chromogenic substrate that changes color responsive to the presence of PC-PLC enzymes, and an ingredient that promotes the expression of the PC-PLC enzyme. In a preferred embodiment, the medium includes a second chromogenic substrate that changes color responsive to the presence of PI-PLC enzymes and an ingredient that promotes the expression of the PI-PLC enzyme.

[0001] The present invention relates to the rapid identification ofBacillus anthrasis, Bacillus cereus, and Bacillus thuringiensis using achromogenic plating medium. It also relates to the differentiation ofBacillus anthrasis from Bacillus cereus, and Bacillus thuringiensis.

BACKGROUND OF THE INVENTION

[0002] Although B. anthrasis (anthrax), B. cereus (foodbornegastrointestinal disease), and B. thuringiensis (biological pesticide)produce a variety of pathological effects, the three bacilli are relatedgenetically with some authors placing these organisms as subspecies ofthe group Bacillus cereus (Turnbull, P.C.B.1999. Definitiveidentification of Bacillus anthrasis-a review, Journal of AppliedMicrobiology, Vol. 87 pages 237-240; Helgason, E. et al. 2000. Bacillusanthrasis, Bacillus cereus, and Bacillus thuringiensis-one species onthe basis of genetic evidence, Applied and Environmental Microbiology,Vol. 66 pages 2627-2630). An easy and rapid separation of these threebacterial strains is important for determining the causative agent of apathological effect, especially with regards to the potential use of B.anthrasis as a biological weapon.

[0003] Traditionally, Bacillus cereus/Bacillus thuringiensis have beenpresumptively isolated from a variety of sources including foods and theenvironment using mannitol egg yolk polymyxin agar (MYP) dependent onexpression of lecithinase activity, fermentation of mannitol andresistance to polymyxin (Compendium of Methods for the MicrobiologicalExamination of Foods, 1992, Chapter 35, American Public HealthAssociation). With the shortcoming of MYP involving frequent falsepositive and negative reactions and coalescing of colonies causingdifficulty in colony enumeration, in 2001 a plating medium using5-bromo-4-chloro-3-indoxyl myo-inositol-l-phosphate to detectphosphatidylinositol˜specific phospholipase C (PI-PLC) in B. cereusi/B.thuringiensis producing turquoise colonies was developed and patented(Peng, H. et al. 2001. Isolation and enumeration of Bacillus cereus fromfoods on a novel chromogenic plating medium, Food Microbiology, Vol. 18pages 231-238; Restaino, L. 2001. Plating media for the presumptiveidentification of Bacillus cereus and Bacillus thuringiensis, U.S. Pat.No. 6,284,517). For B. anthrasis, blood agar containing polymyxin B withincubation at 37° C. for 24 hours has traditionally been used resultingin a large percentage of false positive isolates.

[0004] Although B. cereus, B. thuringiensis; and B. anthrasis producePI-PLC, the molecular weight of this enzyme is different in B. anthrasiscompared with the enzyme produced by the other two bacilli indicating adifferent mechanism of action (Guttmann, D. M. and D. J. Ellar. 2000.Phenotypic and genotypic comparisons of 23 strains from the Bacilluscereus complex for a selection of known and putative B. thuringiensisvirulence factors, FEMS Microbiology Letters, Vol. 188 pages 7-13). Thisreaction can be demonstrated on plating medium containing thechromogenic substrate5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate where afterincubation B. cereus and B. thuringiensis produce turquoise colonies andB. anthrasis yield white colonies. However, phosphatidylcholine-specificphospholipase C (PC-PLC) enzyme is identical in B. cereus, B.thuringiensis and B. anthrasis, but the rate of production is slower forB. anthrasis (Guttmann, D. M. and D. J. Ellar. 2000. Phenotypic andgenotypic comparisons of 23 strains from the Bacillus cereus complex fora selection of known and putative B. thuringiensis virulence factors,FEMS Microbiology Letters, Vol. 188 pages 7-13).

SUMMARY OF THE INVENTION

[0005] It is a principle object of the present invention to provide asingle plating medium with a chromogenic system for the presumptiveidentification of Bacillus cereus, Bacillus thuringiensis and Bacillusanthrasis from a mixed sample. It is also an object of this invention todifferentiate Bacillus anthrasis from Bacillus cereus and Bacillusthuringiensis.

[0006] The inventor realizes that when B. cereus, B. thuringiensis, andB. anthrasis are inoculated in a growth medium and allowed to incubateat an optimal temperature for a required length of time, these bacterialstrains will produce identical PC-PLC, whereas, the PI-PLC from B.anthrasis will differ from the PI-PLC from the other two bacilli.Therefore, it is an object of the present invention to produce a platingmedium for the presumptive isolation of B. cereus, B. thuringiensis, andB. anthrasis that has at least one chromogenic substrate for theidentification of PI-PLC and/or PC-PLC. The inventor realizes that achromogenic substrate (i.e.,5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate) for PI-PLC willidentify this enzyme in B. cereus and B. thuringiensis—(produceturquoise colonies) but not B. anthrasis (produce white to creamcolonies) and it is the purpose of this invention to use a chromogenicsubstrate (i.e., 5-bromo-6-chloro-3-indoxyl-choline-phosphate) forPC-PLC alone or in conjunction with a chromogenic substrate for PI-PLCto differentiate B. anthrasis from B. cereus and B. thuringiensis.

[0007] The inventor has found that the enzymes PI-PLC and PC-PLC producelittle reaction to the substrates in the absence of an ingredient thatpromotes the expression of these enzymes. Hence, a plating mediumaccording to the present invention must comprise a nutrient base thatpromotes the growth of B. cereus, B. thuringiensis, and B. anthrasisunder incubating conditions, at least one chromogenic substratesdetecting PC-PLC with or without at least one chromogenic substratesidentifying PI-PLC, and at least one ingredient that promotes theexpression of the enzymes reacting with the chromogenic substrates.

[0008] In practice, the plating medium according to the presentinvention comprises (1) a nutrient medium that promotes the growth of B.cereus, B. thuringiensis, and B. anthrasis under incubating conditions,(2) at least one ingredient that promotes repair of injured bacillicells under incubating conditions, (3) at least one ingredient thatinhibits the growth of most bacilli other than B. cerceus, B.thuringiensis, and B. anthrasis and other related and unrelated bacteriaunder incubating conditions, (4) at least one ingredient that inhibitsthe growth of yeasts and molds under incubating conditions, (5) at leastone chromogenic substrate detecting PC-PLC with or without at least onechromogenic substrate identifying PI-PLC, (6) at least one ingredientthat promotes the expression of the enzymes reacting with thechromogenic substrates, and (7) at least one ingredient that solidifiesthe mixture.

DETAILED DESCRIPTION OF THE INVENTION

[0009] It is necessary that B. cereus, B. thuringiensis, and B.anthrasis consume nutrients and grow in order for the bacteria tosecrete the sought after enzymes, therefore, the plating medium musthave a rich nutrient base. In order to promote the growth of the soughtafter bacterial strains, the plating medium of the present inventionincludes one or more of the ingredients casein digest, soytone, proteosepeptone, Lab Lemco (meat extract) powder, and yeast extract. In thepreferred medium described throughout this specification, casein digest,Lab Lemco powder and soytone are in the plating medium and form thenutrient base.

[0010] The preferred plating medium includes sodium pyruvate tofacilitate the repair of injured bacilli cells.

[0011] In any selective plating medium, the growth of bacteria cellsother than the sought after bacterial species complicates or can confusethe reading of the plates; therefore, it is desirable to inhibit thegrowth of bacterial species other than the desired bacterial species.The medium of the present invention must suppress most bacteria andBacillus species other than B. cereus, B. thuringiensis, and B.anthrasis. For this purpose, the media of the present inventionpreferably contain one or more of the ingredients: lithium chloride,ceftazidime pentahydrate, polymyxin B sulfate, third or fourthgeneration cephalosporins, and moxalactam. The preferred plating mediumcontains lithium chloride, ceftazidime pentahydrate, and polymyxin Bsulfate. Also, the preferred medium contains cycloheximide to inhibitthe growth of yeasts and molds.

[0012] In the preferred embodimment, the chromogenic substrate thatchanges color responsive to the presence of PC-PLC is5-bromo-4-chloro-3-indoxyl-choline-phosphate. With this chromogenicsubstrate identifying PC-PLC, B. cereus and B. thuringiensis can beisolated from B. anthrasis by discerning differences in the rates ofenzyme expression. Other suitable chromogenic substrates identifyingPC-PLC are 3-Indoxyl-choline phosphate,5-Bromo-6-chloro-3-indoxyl-choline phosphate, 6-Chloro-3-indoxyl-cholinephosphate, 5-Iodo-3-indoxyl -choline phosphate, N-Methylindoxyl-cholinephosphate, 2-Nitrophenyl-choline phosphate, 3-Nitrophenyl-cholinephosphate, and 4-Nitrophenyl-choline phosphate.

[0013] In addition, a second chromogenic substrate is preferably addedto the plating medium identifying the PI-PLC enzyme. With twochromogenic substrates identifying PC-PLC and PI-PLC incorporated in thepreferred plating medium, after incubation, the B. cereus and B.thuringiensis colonies will display a third color resulting fromenzymatic reactions on the two chromogens; whereas, the color of the B.anthrasis colonies will result from the chromogenic substrateidentifying PC-PLC only.

[0014] Ingredients that promote the expression of the PC-PLC and PI-PLCenzymes in the plating medium are bovine serum, silicates, Tween 80(polyoxyethylenesorbitan monooleate), other variations of Tweenincluding polyoxyethylenesorbitan tristearate, polyoxyethylenesorbitanmonostearate, polyoxyethylenesorbitan monopalmitate,polyoxyethylenesorbitan monolaurate (Tween 21 and 20) and other similarnonionic detergents, and manganese containing compounds, namely,manganese chloride, manganese acetate tetrahydrate, manganese nitratetetrahydrate, and manganese sulfate monohydrate. Table 1 presents thesalts of various divalent cations versus the expression of PC-PLC in thepresence of 5-bromo-4-chloro-3-indoxyl-choline-phosphate. Afterincubation at 37° C., the only divalent cation that produced blue orturquoise colonies was manganese chloride. After 48 hours, B cereus andB. thuringiensis produced turquoise colonies with a narrow rim, whereas,B. anthrasis yielded a cream color colony with a blue dot in the centerof the colony. In the preferred embodiment, the ingredients are bovineserum, Tween 80 and manganese chloride. TABLE 1 EFFECT OF VARIOUSMINERAL CATIONS ON THE EXPRESSION OF PHOSPHATIDYLCHOLINE-SPECIFICPHOSPHOLIPASE C IN BACILLUS CEREUS, BACILLUS THURINGIENSJS AND BACILLUSANTHRASIS INCUBATED AT 37° C. FOR 24 AND 48 HOURS Bacillus anthrasisBacillus thuringiensis Bacillus cereus AMES-RIID and ATCC 10792 ATCC14579 ANR-1 Colonial Colonial Colonial Mineral Morphologies MorphologiesMorphologies Cations 24 hours 48 hours 24 hours 48 hours 24 hours 48hours No added Large Large Large Large Small Medium cations cream creamcream cream cream cream colored colored colored colored colored colored0.1% Turquoise Turquoise Turquoise Turquoise Small Cream Manganese withwith with with cream with chloride white rim white rim white rim whiterim colored blue center 0.12% Large Large Large Large Small MediumMagnesium cream cream cream cream cream cream sulfate colored coloredcolored colored colored colored 0.074% Calcium Large Large Large LargeSmall Medium chloride cream cream cream cream cream cream coloredcolored colored colored colored colored 0.015% Zinc Large Large LargeLarge Small Medium sulfate cream cream cream cream cream cream coloredcolored colored colored colored colored Small Small Small Small No No0.078% Cupric colorless colorless colorless colorless growth growthsulfate to small to small colorless colorless

[0015] An ingredient must be added to the mixture to solidify themixture. In the preferred composition, this ingredient is agar.

[0016] The formula for the preferred embodiment of the plating medium ispresent in Table 2. TABLE 2 FORMULA FOR THE PREFERRED EMBODIMENT OF THEPLATING MEDIUM Chemical Supplier Grams/liter Casein Digest Difco 15.00Lab Lemco Powder Oxoid 5.00 Soytone Difco 5.00 Sodium pyruvate Biosynth10.00 Tween 80 — 0.5 (polyoxyethylenesorbitan monooleate Sodium chloride— 5.0 Manganese chloride — At least 1.0 grams tetrahydrate Cycloheximide— 0.20 Lithium chloride Sigma 2.00 Agar Difco 15.00 Bovine serum 82-067Serologicals 3.20 Ceftazidime pentahydrate Glaxo Wellcome 0.045-bromo-4-chloro-3 Biosynth 0.32 indoxyl-choline phosphate or otherchromogenic or fluorogenic substrates* Polymyxin B sulfate Sigma 100,000units

[0017] Prior to the preparation of the selective/differential platingmedium, all the heat resistant ingredients are mixed into a vesselcontaining 970 ml of deionized/distilled water. The mixture should bewarmed slightly and stirred to dissolve any clumps and powder. The pH ofthe mixture should be recorded within a range of 6.80 to 7.20. Theplating medium is sterilized at 121-124° C. for 15 minutes. Aftersterilization, the medium is cooled in a water bath at 50° C.Thereafter, one at a time, the heat sensitive ingredients, including thechromogenic substrate(s), bovine serum, ceftazidime pentahydrate, andpolymyxin B sulfate, are added to 30 ml of deionized/distilled water anddissolved, hereafter referred to as the supplement. The supplement isfilter-sterilized and poured into the cooled sterile plating medium. Thecompleted medium is swirled and the composition is placed in Petridishes and stored under proper conditions overnight. The final pH of theplating medium is 6.80 to 7.20. The plating medium is stable up to 60days stored in a plastic sleeve at 4-8° C.

EXAMPLE I

[0018] The bacterial strains indicated in Table 3 were applied to thePetri dishes referred to above (using only the5-bromo-4-chloro-3-indoxyl-choline phosphate chromogenic substrate), andincubated at 35-37° C. for a period of 48 hours. Thereafter, thesurfaces of the plating medium in the Petri dishes were observed, andproduced the following results presented in Table 3. TABLE 3 COLONIALMORPHOLOGIES OF VARIOUS BACTERIAL STRAINS ON THE PLATING MEDIUM AT35-37° C. FOR 48 HOURS Number Bacteria of strains Colonial MorphologiesBacillus cereus 7 Teal flat dull colonies with white rim Bacillusthuringiensis 5 Teal flat dull colonies with white rim Bacillusanthrasis 2 Cream flat dull colonies with blue dot in the centerBacillus circulans 1 White domed dull colonies Bacillus megaterium,Bacillus 1 strain each No growth licheniformis, Bacillus subtilis,Bacillus brevis, Bacillus lentus, Bacillus pumilus, Bacillus spaericus,Bacillus mycoides and, Bacillus insolitus PaeniBacillus macerans, and 1strain each No growth PaeniBacillus polymyxa Listeria monocytogenes 3 Nogrowth to white pinpoint domed colonies Listeria ivanovii, Listeriainnocua, 1 strain each White domed colonies; Listeria seeligeri, andListeria pinpoint to <1 mm welshmeri Enterococcus faecium 4 No growth towhite domed colonies; pinpoint Enterococcus faecalis 2 No growth towhite domed colonies; pinpoint Enterococcus avium 3 No growthStaphylococcus aureus 5 No growth Micrococcus sp., Pediococcus 1 straineach No growth cerevisiae, Staphylococcus epidermidis, andStaphylococcus saprophyticus Gram negative species* 7 No growth

[0019] From Table 3 it is obvious that B. cereus/B. thuringiensisproduce a teal colored colony that is easily distinguished from the B.anthrasis colonies which are cream flat dull with a blue dot center. Thegrowth of other PC-PLC producing bacteria are inhibited by the platingmedium, and are eliminated as potential false positives. Any other nonproducing PC-PLC bacteria growing on the plating medium will form whitecolored colonies.

EXAMPLE 2

[0020] A second set of Petri dishes were prepared as indicated above,except a second chromogenic substrate(5-bromo-4-chloro-3-indoxyl-myo-inositol-l-phosphate) was added to themixture with the first chromogenic substrate. The bacterial strainsindicated in Table 3 were applied to the second set of Petri dishes(using both the 5-bromo-4-chloro-3-indoxyl-choline phosphate chromogenicsubstrate and the 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphatesubstrate), and incubated at 35-37° C. for a period of 48 hours.Thereafter, the surfaces of the plating medium in the second set ofPetri dishes were observed. Bacillus anthrasis colonies were observed tobe substantially the same as set forth in Table 3, namely, cream flatdull colonies with a blue dot in the center. However, the Bacilluscereus and Bacillus thuringiensis colonies appeared with a color that isa blend of the colors of the two substrates (blue and turquoise,respectively), namely, bluish-turquoise.

[0021] Although variations in the plating medium of the preferredembodiment are set forth above, other variations will become apparent tothose skilled in the art. It is therefore intended that this inventionbe not limited to the foregoing specification, but rather only to theappended claims.

The invention claimed is:
 1. A plating medium for the presumptiveidentification of Bacillus cereus, Bacillus thuringiensis and/orBacillus anthrasis comprising a nutrient base to facilitate growth ofBacillus cereus, Bacillus thuringiensis and Bacillus anthrasis, at leastone member of a group consisting of a first chromogenic substrate thatchanges color responsive to the presence of PC-PLC enzymes, and a secondchromogenic substrate that changes color responsive to the presence ofPI-PLC enzymes, and at least one ingredient that promotes the expressionof the PC-PLC and PI-PLC enzymes.
 2. A plating medium for thepresumptive identification of Bacillus cereus, Bacillus thuringiensisand/or Bacillus anthrasis comprising claim 1 wherein the medium containsa first chromogenic substrate that changes to a first color responsiveto the presence of PC-PLC enzymes, and a second chromogenic substratethat changes to a second color responsive to the presence of PI-PLCenzymes, the first and second colors being different and blending to athird color that contrasts with the first and second colors.
 3. Aplating medium for the presumptive identification of Bacillus cereus,Bacillus thuringiensis and/or Bacillus anthrasis comprising claim 1 incombination with an ingredient to suppress the growth of bacteria otherthan Bacillus cereus, Bacillus thuringiensis and Bacillus anthrasis. 4.A plating medium for the presumptive identification of Bacillus cereus,Bacillus thuringiensis and/or Bacillus anthrasis comprising claim 3wherein the ingredient to suppress the growth of bacteria other thanBacillus cereus, Bacillus thuringiensis and Bacillus anthrasis is one ormore members of the class lithium chloride, ceftazidime pentahydrate,polymyxin B sulfate, third or fourth generation cephalosporins, andmoxalactam.
 5. A plating medium for the presumptive identification ofBacillus cereus, Bacillus thuringiensis and/or Bacillus anthrasiscomprising claim 1 wherein the first chromogenic substrate that changescolor responsive to the presence of PC-PLC enzymes is a member of thegroup 5-bromo-4-chloro-3-indoxyl-choline phosphate, 3-Indoxyl-cholinephosphate, 5-Bromo-6-chloro-3indoxyl-choline phosphate,6-Chloro-3-indoxyl-choline phosphate, 5-Iodo-3-indoxyl-cholinephosphate, N-Methylindoxyl-choline phosphate, 2-Nitrophenyl-cholinephosphate, 3-Nitrophenyl-choline phosphate, and 4-Nitrophenyl-cholinephosphate.
 6. A plating medium for the presumptive identification ofBacillus cereus, Bacillus thuringiensis and/or Bacillus anthrasiscomprising claim 1 wherein the second chromogenic substrate that changescolor responsive to the presence of PI-PLC enzymes is5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate.
 7. A plating mediumfor the presumptive identification of Bacillus cereus, Bacillusthuringiensis and/or Bacillus anthrasis comprising claim 2 wherein thefirst chromogenic substrate that changes color responsive to thepresence of PC-PLC enzymes is 5-bromo-4-chloro-3-indoxyl-cholinephosphate, and the second chromogenic substrate that changes colorresponsive to the presence of PI-PLC enzymes is5-bromo-4-chloro-3-indoxyl-myo -inositol-1-phosphate.
 8. A platingmedium for the presumptive identification of Bacillus cereus, Bacillusthuringiensis and/or Bacillus anthrasis comprising claim 7 wherein. theingredient in the plating medium that promotes the expression of thePC-PLC and PI-PLC enzymes is a member of the group bovine serum,silicates, Tween, manganese chloride, manganese acetate tetrahydrate,manganese nitrate tetrahydrate, and manganese sulfate monohydrate.
 9. Aplating medium for the presumptive identification of Bacillus cereus,Bacillus thuringiensis and/or Bacillus anthrasis comprising claim 1wherein the nutrient base to facilitate growth of Bacillus cereus,Bacillus thuringiensis and Bacillus anthrasis is at least one member ofthe group casein digest, soytone, proteose peptone, meat extract powder,and yeast extract.
 10. A plating medium for the presumptiveidentification of Bacillus cereus, Bacillus thuringiensis and/orBacillus anthrasis comprising claim 1 in combination with at least oneingredient to repair injured bacilli cells.
 11. A plating medium for thepresumptive identification of Bacillus cereus, Bacillus thuringiensisand/or Bacillus anthrasis comprising claim 10 wherein the ingredient torepair injured bacilli cells is sodium pyruvate.
 12. A plating mediumfor the presumptive identification of Bacillus cereus, Bacillusthuringiensis and/or Bacillus anthrasis comprising claim 1 incombination with at least one ingredient to solidify the mixture.
 13. Aplating medium for the presumptive identification of Bacillus cereus,Bacillus thuringiensis and/or Bacillus anthrasis comprising: a nutrientbase comprising one or more members of the group casein digest, soytone,proteose peptone, meat extract powder, and yeast extract; a firstchromogenic substrate that changes to a first color responsive to thepresence of PC-PLC enzymes comprising 5-bromo-4-chloro-3-indoxyl-cholinephosphate; a second chromogenic substrate that changes to a second colorresponsive to the presence of PI-PLC enzymes comprising5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate; the first andsecond colors blending to a third color that contrasts with the firstand second colors, an ingredient that promotes the expression of thePC-PLC and PI-PLC enzymes comprising one or more members of the classbovine serum, silicates, and manganese chloride; sodium pyruvate; andagar to solidify the mixture.
 14. A plating medium for the presumptiveidentification of Bacillus cereus, Bacillus thuringiensis and/orBacillus anthrasis comprising a nutrient base to facilitate growth ofBacillus cereus, Bacillus thuringiensis and/or Bacillus anthrasis, achromogenic substrate that changes color responsive to the presence ofPC-PLC enzymes, and an ingredient that promotes the expression of PC-PLCenzymes.
 15. A plating medium for the presumptive identification ofBacillus cereus, Bacillus thuringiensis and/or Bacillus anthrasiscomprising claim 14 wherein the chromogenic substrate is5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate, and the ingredientthat promotes the expression of PC-PLC enzymes is a member of the groupbovine serum, silicates, and manganese chloride and other manganesecontaining compounds.
 16. A plating medium for the presumptiveidentification of Bacillus cereus, Bacillus thuringiensis and/orBacillus anthrasis comprising a nutrient base to facilitate growth ofBacillus cereus, Bacillus thuringiensis and Bacillus anthrasis, achromogenic substrate that changes color responsive to the presence ofPI-PLC enzymes, and an ingredient that promotes the expression of PI-PLCenzymes.
 17. A plating medium for the presumptive identification ofBacillus cereus, Bacillus thuringiensis and/or Bacillus anthrasiscomprising claim 16 wherein the chromogenic substrate is5-bromo-4-chloro-3-indoxylmyo-inositol-1-phosphate, and the ingredientthat promotes the expression of PI-PLC enzymes is a member of the groupbovine serum, silicates, Tween, manganese chloride, manganese acetatetetrahydrate, manganese nitrate tetrahydrate, and manganese sulfatemonohydrate.
 18. A plating medium for the presumptive identification ofBacillus cereus, Bacillus thuringiensis and/or Bacillus anthrasiscomprising claim 6 wherein. the ingredient in the plating medium thatpromotes the expression of the PC-PLC and PI-PLC enzymes is a member ofthe group bovine serum, silicates, Tween, manganese chloride, manganeseacetate tetrahydrate, manganese nitrate tetrahydrate, and manganesesulfate monohydrate.